Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.
Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.
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利用 3-吲哚丙酰基/丙烯酰基-CoA 作为发色底物类似物对中链脂肪酰基-CoA 脱氢酶进行机理研究。
DOI：
10.1021/bi00158a020
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
D. Srivastava
中科院分区：
文献类型：
--
作者：
J. Johnson;Z. X. Wang;D. Srivastava
辅酶A衍生物3-吲哚丙酰辅酶A（IPCoA）作为中链脂肪酰辅酶A脱氢酶（MCAD）催化反应的有效假底物。反应产物反式-3-吲哚丙烯酰基-CoA（IACoA）表现出特征性紫外-可见吸收光谱，λ max = 367 nm，λ 367 = 26，500 M-1 cm-1。IACoA的发色性质使我们能够测量底物到产物的直接转化（在367 nm处），而无需求助于酶结合黄素或耦合电子受体的吸收信号，以及探测酶位点环境。IACoA与中链脂肪酰辅酶A脱氢酶（MCAD）-FAD的相互作用的特征在于在490、417和355 nm处产生的吸收峰（混合物减去单个组分的光谱）。这些吸收峰的大小随着缓冲介质的pH值降低而增加。MCAD-FAD与IACoA相互作用的瞬时动力学分析表明，酶-IACoA复合物的形成分两步进行。第一步（快速）涉及E-IACoA碰撞复合物的形成，在第二步（缓慢）中，E-IACoA碰撞复合物[分子式：见正文]被异构化（伴随蛋白质结构的变化）为E*-IACoA复合物。我们研究了pH对Kc、K2和K-2的影响。虽然Kc几乎没有显示对pH的依赖性（在pH 6.0和9.5之间的2倍变化），k2和k-2显示对pH的强烈依赖性。k2和k-2都表现出对缓冲介质的pH的S形依赖性，pKa分别为7.53和8.30。根据本文提出的模型，7.53的pKa代表参与与E-IACoA碰撞复合物内的IACoA相互作用的酶位点基团。在碰撞复合物异构化后，该pKa被扰动至8.30。K2和K-2的pH依赖性变化使得E-IACoA和E*-IACoA之间的平衡分配有利于后者复合物（约20倍）。光谱，动力学，酶-IACoA复合物的热力学性质使我们能够描绘出E-IACoA异构化的微观途径（推测通过蛋白质构象变化）与质子平衡步骤偶联。
The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.
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猪肾酰基辅酶A脱氢酶与电子传递黄素蛋白之间的间黄素氧化还原反应。
影响因子：
2.9
作者：
Gorelick,RJ;Schopfer,LM;Ballou,DP;Massey,V;Thorpe,C
通讯作者：
Thorpe,C
一般酰基辅酶A脱氢酶的酶激活抑制剂、替代底物和死端抑制剂。
DOI：
--
发表时间：
1980
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Frerman,FE;Miziorko,HM;Beckmann,JD
通讯作者：
Beckmann,JD
影响因子：
2.9
作者：
GHISLA, S;THORPE, C;MASSEY, V
通讯作者：
MASSEY, V
酰基辅酶 A 底物和类似物与猪肾中链酰基辅酶 A 脱氢酶的相互作用。
影响因子：
2.9
作者：
Powell,PJ;Lau,SM;Killian,D;Thorpe,C
通讯作者：
Thorpe,C
来自猪肾的中链酰基辅酶 A 脱氢酶具有固有的烯酰辅酶 A 水合酶活性。
影响因子：
2.9
作者：
Lau,SM;Powell,P;Buettner,H;Ghisla,S;Thorpe,C
通讯作者：
Thorpe,C